1. Sonicate crosslinked DNA to fragments sizes of 200-1000 base pairs. Keep samples ice cold to prevent denaturing of chromatin. Conditions for fragmenting must be empirically derived, and vary depending on equipment, cell type, cell density, and cross-linking efficiency.
2. Centrifuge samples to remove debris at 4C for 10 minutes at 12,500 x g. Remove supernatant and transfer to a new tube. Discard pellet. Set aside 75 ul of sample for input fraction, which will not go through the subsequent IP steps. The remaining sample can be moved into 75 ul aliquots, each of which is sufficient for a single IP. Although it is preferable to proceed directly to the following steps, sheared chromatin can now be frozen at -80C for up to 1 month.
a. Optional: Test the efficiency of the shearing by running 5-10 ul of sample on a 2% agarose gel after reversal of crosslinking, RNase treatment (0.5 mg/ml) and proteinase K treatment (0.1 ug/ul) as described below.